p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation - PubMed (original) (raw)

p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation

J Xu et al. Mol Cell Biol. 1999 Jan.

Abstract

The proliferating cell nuclear antigen (PCNA) is a highly conserved cellular protein that functions both in DNA replication and in DNA repair. Exposure of a rat embryo fibroblast cell line (CREF cells) to gamma radiation induced simultaneous expression of PCNA with the p53 tumor suppressor protein and the cyclin-dependent kinase inhibitor p21(WAF1/Cip1). PCNA mRNA levels transiently increased in serum-starved cells exposed to ionizing radiation, an observation suggesting that the radiation-associated increase in PCNA expression could be dissociated from cell cycle progression. Irradiation of CREF cells activated a transiently expressed PCNA promoter chloramphenicol acetyltransferase construct through p53 binding sequences via a mechanism blocked by a dominant negative mutant p53. Electrophoretic mobility shift assays with nuclear extracts prepared from irradiated CREF cells produced four p53-specific DNA-protein complexes with the PCNA p53 binding site. Addition of monoclonal antibody PAb421 (p53-specific) or AC238 (specific to the transcriptional coactivator p300/CREB binding protein) to the mobility shift assay distinguished different forms of p53 that changed in relative abundance with time after irradiation. These findings suggest a complex cellular response to DNA damage in which p53 transiently activates expression of PCNA for the purpose of limited DNA repair. In a population of nongrowing cells with diminished PCNA levels, this pathway may be crucial to survival following DNA damage.

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Figures

FIG. 1

Levels of p53, p21, and PCNA in CREF cells at various times postirradiation. Asynchronously growing subconfluent cells were exposed to 12 Gy of IR, and whole-cell lysates were prepared at the indicated times postexposure. Equal amounts of protein from each lysate were fractionated in polyacrylamide gels, which were probed by immunoblotting with specific antibodies to p53 (top), p21 (middle), and PCNA (bottom). Levels of these proteins in unirradiated CREF cells (Un; lane 1) and at 1, 3, 8, and 24 h postirradiation (lanes 2 to 5) are shown. The slight decrease in PCNA expression at 1 h postirradiation was not reproducible.

FIG. 2

IR increases cellular levels of PCNA. Confluent cultures of CREF cells were released from growth arrest by replating at 70% confluence. Six hours after replating, the cells were mock irradiated or exposed to 12 Gys of γ radiation. Cell lysates were prepared in radioimmunoprecipitation assay buffer from the unirradiated and irradiated cells, and equal amounts of protein from each lysate were assessed for PCNA levels by immunoblotting. Top and bottom panels show immunoblots indicating PCNA levels in irradiated and unirradiated CREF cells, respectively, at the indicated times postexposure.

FIG. 3

PCNA mRNA levels in irradiated CREF cells. Total cell RNA was prepared from unirradiated (lane 1) or irradiated (lanes 2 to 5) CREF cells kept in DMEM containing 0.1% FBS at the indicated times postexposure to 12 Gys of IR. Each sample (30 μg) was fractionated in a formaldehyde-agarose gel that was subsequently transferred to a polyvinylidene difluoride membrane. The blot was probed by hybridization to radioactive cDNA probes specific for β-actin and PCNA. The hybridized filter was exposed to X-ray film with an intensifying screen for 48 h.

FIG. 4

Transient transfection and irradiation in CREF cells. (A) Diagram of PCNA-CAT reporter constructs and experimental protocol. The position of the p53 binding site is indicated by the stippled box in the −249 construct. NT, nucleotides. (B) γ irradiation activates the PCNA promoter via the wild-type p53 binding site. Twenty-four hours postirradiation, the cells transfected with the −249 or −213 construct (A) were harvested, and CAT activity was determined for equal amounts of protein from each lysate. The graph shows the fold change (average ± standard error for three different experiments performed in duplicate) in CAT activity versus the indicated dose of IR (relative to unirradiated cells, 0 Gy) for transfected cells. (C) A dominant negative mutant p53 (DMp53) prevents IR-induced activation of the PCNA promoter. The protocol was the same as for panel B except that the CREF cells were cotransfected with pCMV-DMp53 and the −249PCNA-CAT or −213PCNA-CAT construct prior to irradiation. The results shown are averages of two experiments performed in duplicate.

FIG. 5

Binding specificity of complexes formed in EMSAs with nuclear extracts prepared from irradiated CREF cells. (A) Binding to the PCNA p53 binding site. A double-stranded oligonucleotide corresponding to the PCNA p53 binding site (PCNA) was used as the radiolabeled probe. Double-stranded oligonucleotides corresponding to the p53 binding site in the WAF1 gene (p21), the ribosomal gene cluster (RGC), or a mutated version of the PCNA site (MtPCNA) that fails to bind p53 (70) were used as unlabeled competitors in the EMSA. Lanes 1 and 2 show the radiolabeled probe without extract and with extract prepared from CREF cells 3 h postirradiation, respectively. Experimental conditions for lanes 3 to 10 were identical to those for lane 2 except that the indicated competitors were included in the binding mix at either 40- or 80-fold excess compared to labeled probe. The four specific complexes that form in the assay are designated C1 to C4. The gel was overexposed (more than 48 h) to reveal the oligonucleotide competition for all four complexes. The detection of the relatively minor C4 complex varied between experiments. (B) Binding to the p53 binding site of the WAF1 gene. Details are as for panel A except that a double-stranded oligonucleotide corresponding to the p21_WAF1_ p53 binding site was used as the radiolabeled probe. Complexes B1 through B4 comigrate with complexes C1 through C4 formed with the PCNA probe in panel A (not shown). Lane 1, WAF1 probe without extract; lane 2, WAF1 probe with nuclear extract prepared from CREF cells at 3 h postirradiation.

FIG. 6

DNA-protein complex formation with the PCNA p53 binding site varies with time postirradiation. A radiolabeled oligonucleotide corresponding to the PCNA p53 binding site was used as the probe with nuclear extracts prepared from uniradiated cells (lane 2) or irradiated cells at the indicated times (hours) postirradiation (lanes 3 to 6). The pattern of the probe without extract is shown in lane 1. Lanes 7 to 11 are identical to lanes 2 to 6 except that 1 μl of p53-specific monoclonal antibody PAb421 was added in each binding mix. Complexes C1 to C4 are designated as in Fig. 5. The arrow indicates the new, slower-migrating complex formed in the presence of PAb421. Of a variety of antibodies tested, no other antibody produced a band with the mobility of the arrow. The faint band below C3 is not reproducible between experiments. The gel was exposed to X-ray film for 15 to 20 h.

FIG. 7

Binding of p300/CBP to p53 in irradiated CREF cells. (A) Specificity of anti-p300/CBP antibody. Binding to an oligonucleotide corresponding to the radiolabeled PCNA p53 binding site was assessed by EMSA with equal amounts of nuclear extract prepared from CREF cells 3 h after irradiation. The assays were without antibody (lane 1), with 2 μl of anti-p300/CBP AC238 ascites fluid (lane 2), with 4 μl of anti-MDM-2 2A10 monoclonal supernatant (lane 3), or with 2 μl of anti-PCNA 19F4 (lane 4). ∗, a new complex generated by adding the p300/CBP-specific antibody. (B) Binding to p300/CBP varies with time postirradiation. Nuclear extracts were isolated at the indicated times (h) postirradiation. Equal amounts of protein from each extract were evaluated by EMSA with an oligonucleotide corresponding to the PCNA p53 binding site. The extracts from unirradiated (lane 1) and irradiated (lane 2 to 5) cells were preincubated with 2 μl of AC238 (anti-p300/CBP) before the addition of probe.

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p53-mediated regulation of proliferating cell nuclear antigen expression in cells exposed to ionizing radiation - PubMed (original) (raw)